Nerve Growth Factor (NGF) and adenosine 3'-5' monophosphate (cAMP) have been shown to promote in vitro neuronal maturation. Our recent finding that bovine brain gangliosides also enhance in vitro metabolic activity and neurite elongation suggests that the mechanism(s) by which NGF, gangliosides and cAMP stimulate neuronal differentiation may be interrelated. To elucidate some of the mechanisms by which these agents (promote" neurite extension, the three dimensional organization and activity of actin, myosin, tubulin and troponin-C will be studied with: (1) immunocytochemistry, (2) Nomarski and interference reflexion microscopy and (3) transmission and scanning electron microscopy. Our immunofluorescence studies have shown the presence of myosin-rich filaments in regions known to contain actin filaments; an arrangement suggesting an active role for myosin in neurite extension. We have shown that diazepam specifically inhibits myosin synthesis in cultured neurons. Diazepam, cytochalasin D and colcemid will be used to probe the role of myosin, actin and tubulin in neurite elongation. The contributory roles of the contractile proteins in ganglioside-, NGF- and cAMP-induced elongation will be studied with agents which affect their integrity and/or functional activity. Gangliosides and cAMP will also be used to enhance in vivo neuronal regeneration. These studies will provide important information on the mechanisms underlying development and regeneration of the nervous system.